Correct promoter control is needed for trafficking of the ring-infected erythrocyte surface antigen to the host cytosol in transfected malaria parasites

Abstract

Following invasion of human erythrocytes, the malaria parasite, Plasmodium falciparum, exports proteins beyond the confines of its own plasma membrane to modify the properties of the host red cell membrane. These modifications are critical to the pathogenesis of malaria. Analysis of the P. falciparum genome sequence has identified a large number of molecules with putative atypical signal sequences. The signals remain poorly characterized; however, a number of molecules with these motifs localize to the host erythrocyte. To examine the role of these atypical signal sequences in the export of parasite proteins, we have generated transfected parasites expressing a chimeric protein comprising the N-terminal region of the P. falciparum ring-infected erythrocyte surface antigen (RESA) appended to green fluorescent protein (GFP). This N-terminal region contains a hydrophobic stretch of amino acids that is presumed to act as a noncanonical secretory signal sequence. Modulation of the timing of transgene expression demonstrates that trafficking of malaria proteins into the host erythrocyte is dependent on both the presence of an appropriate transport signal and the timing of expression. Transgene expression under the control of a trophozoite-specific promoter mistargets the chimeric molecule to the parasitophorous vacuole surrounding the parasite. However, expression of RESA-GFP in schizont stages, under the control of the RESA promoter, enables correct trafficking of a population of the chimeric protein to the host erythrocyte.

FIG. 1.

Organization of the RESA gene and transfection constructs. (A) Schematic diagram of the pHH2 and pRESA5′ constructs, showing insertion of RESA_1-117 upstream of the GFP coding region. (B) Schematic diagram of the RESA gene. Exon 1 encodes residues 1 to 65, including a recessed putative hydrophobic signal sequence. Exon 2 encodes residues 66 to 1073, including the 5′ and 3′ acidic repeats, a DnaJ domain, and a spectrin-binding domain. (C) The N-terminal 117-amino-acid fragment of RESA appended to GFP (RESA_1-117-GFP). The hydrophobic core of the putative signal sequence is double underlined. The putative processing site for the plasmodial signal peptidase in the ER is shown with an arrowhead, and the GFP tag is shown in italics.

FIG. 2.

Transgene expression in parasites transfected with the RESA_1-117-GFP constructs. (A) RNA was prepared from tightly synchronized cultures (∼4-h window) collected at 4-h intervals, separated on agarose gels, and transferred to nylon membranes. The membranes were hybridized to α-[³²P]dATP-labeled GFP or RESA DNA fragments and exposed to light-sensitive film. The films were scanned, and the levels of hybridization of the GFP probe to the RESA_1-117-GFP_Hsp86 sample (diamonds) and the RESA_1-117-GFP_RESA sample (squares) and of the RESA probe to a sample prepared from untransfected 3D7 parasites (triangles) were quantitated by image analysis. (B) Saponin-lysed asynchronous parasite cultures (10% parasitemia) were subjected to SDS-PAGE (12.5%), transferred to nitrocellulose, and probed with anti-GFP antibody (Roche; 1/1,000 dilution) and anti-RESA (MAb 28/2; 1/1,000 dilution). Lane a, untransfected parasites; lane b, RESA_1-117-GFP_Hsp86 transfectants; lane c, RESA_1-117-GFP_RESA transfectants. The small arrows indicate the 32/31-kDa species, while the larger arrow indicates the 27-kDa species. (C) Samples were taken at 4-h intervals from tightly synchronous parasite cultures (10% parasitemia), frozen, and thawed, and equal amounts of cells were subjected to Western analysis and probed with anti-GFP antibody (Roche; 1/1,000 dilution) or anti-Hsp70 (1/4,000 dilution). Arrowhead, 43-kDa band (unprocessed protein); small arrows, 32/31-kDa doublet; arrow, 27-kDa band (degradation product).

FIG. 3.

Expression of the RESA_1-117-GFP chimeric proteins at different stages of the intraerythrocytic life cycle of P. falciparum. The first image in each set represents the fluorescence signal from the GFP chimeric protein, the second is a bright-field or phase image, and the third shows an overlay of these images. Left panels, RESA_1-117-GFP_Hsp86. Ring and early trophozoite stage parasites (B and C) show PV labeling, with the fluorescence signal resembling a necklace of beads. Some of the PV subcompartments are marked with white arrowheads (B and C). In some cells, GFP accumulates in the food vacuole (D) (red arrowhead). Mature trophozoite stage parasites show labeling mainly within the parasite (D). Occasional extensions or blebs of the PV are observed (white arrows in panels D and E). Schizonts show labeling of the PV surrounding individual merozoites (E). A burst schizont, in which the PV-located GFP has been released, shows a “bunch-of-grapes” pattern with a highly fluorescent central remnant body and a fluorescent structure at the cell periphery (blue arrowhead). Right panels, RESA_1-117-GFP_RESA. The very early stage parasites show a beaded PV pattern (G) (one bead is marked with white arrowhead). As the parasite matures, the cells show bright fluorescence from within the parasite and a weaker fluorescence signal from the erythrocyte cytosol (H to K). In more mature parasites, occasional extensions or blebs of the PV are observed (I) (white arrow). Schizonts show labeling of the PV surrounding individual merozoites (L). Fluorescence from GFP was captured in live cells by using a Zeiss Axioskop 2. Bar, 5 μm. The intensities of the images were adjusted to optimize the fluorescence signal at each parasite stage.

FIG. 4.

Subfractionation of the RESA_1-117-GFP transfectants. Transfectants were grown to trophozoite stage after synchronization, Plasmagel purified (approximately 1 × 10⁷ RESA_1-117-GFP_Hsp86 and 5 × 10⁷ RESA_1-117-GFP_RESA transfectants), solubilized in 4 hemolytic units of SLO or 0.09% saponin, and separated into supernatant (S) and pellet (P) fractions by centrifugation. The supernatant of the saponin fractions contains soluble proteins of the PV and erythrocyte, and the pellet contains the parasite content and all membrane fractions. After SLO lysis, the supernatant contains the soluble contents of the erythrocyte compartment, while the pellet contains the remainder of the proteins. The different fractions were separated by SDS-PAGE (12.5% acrylamide), blotted onto a nitrocellulose membrane, and probed with antibodies recognizing S antigen, PfERC, and GFP. The two left-facing arrowheads indicate the 32- and 27-kDa species. The right-facing arrowhead indicates the 31-kDa species. (A) In RESA_1-117-GFP_Hsp86 transfectants, RESA-GFP is released into the supernatant from cells by saponin but is retained during SLO treatment. (B) In RESA_1-117-GFP_RESA transfectants, RESA-GFP is released from cells by either saponin or SLO treatment. The 31-kDa species appears to be released preferentially.

FIG. 5.

Immunofluorescence microscopy analysis of endogenous RESA and RESA_1-117-GFP in transfected parasitized erythrocytes. Erythrocytes infected with RESA_1-117-GFP_Hsp86 (A) or RESA_1-117-GFP_RESA (B) transfectants at the ring stage (top panels), trophozoite stage (middle panels), or schizont stage (bottom panels) were fixed with methanol (which destroys the intrinsic GFP fluorescence) and labeled with rabbit anti-GFP antiserum followed by an Alexa Fluor 568-conjugated anti-rabbit IgG (red fluorescence) and anti-RESA MAb, followed by fluorescein isothiocyanate-conjugated anti-mouse IgG (green fluorescence). Phase-contrast images are shown in the left panels. Overlays of the green and red fluorescence channels are shown in the right panels. Bar, 5 μm. DAPI, 4′,6′-diamidino-2-phenylindole.

FIG. 6.

Transmission electron microscopy and immunogold labeling of RESA_1-117-GFP transfectants. Sections were probed with rabbit anti-GFP, followed by 15-nm-diameter gold-conjugated anti-rabbit IgG and/or 28/2 anti-RESA MAb, followed by 10-nm-diameter gold-conjugated anti-mouse IgG. (A) Sections through RESA_1-117-GFP_Hsp86 transfectants showing labeling of endogenous RESA at the host membrane and in the PV (arrowheads) (a and b) and associated with structures in the erythrocyte cytosol (b) and in the parasite cytoplasm (c). Larger gold particles representing the RESA_1-117-GFP_Hsp86 product can be seen in the parasite cytosol (arrows) (a and c). (B) Sections through RESA_1-117-GFP_RESA transfectants showing labeling of endogenous RESA at the host membrane (arrowheads) (a and b) and labeling of the RESA_1-117-GFP_RESA product in the parasite cytosol, in the PV, and in the host cell cytosol (arrows). Bars, 100 nm.